The principal objectives of this research project are five-fold: 1) to determine host and viral factors which influence the genetically controlled resistance of inbred mouse strains to street rabies virus (SRV) 2) to determine if an in vivo laboratory model for persistent rabies virus infection can be established with subsequent goals of determining in which cell(s) the virus (genome) is sequestered and whether the virus can be activated to produce clinical disease and death 3) to understand the role of macrophages in rabies virus infections 4) to compare the protective capabilities of recombinant vaccines expressing different or multiple structural proteins of the rabies virus against genetic variants of rabies virus that have been isolated worldwide and 5) to advance our understanding of the complex interaction between microglial cells, lymphocytes and cytokines in the pathogenesis of rabies virus infections. Studies have determined that anti-rabies virus nucleoprotein antisera is protective and inhibits rabies virus replication. Experiments using a recombinant vaccine dually expressing this highly conserved protein and the glycoprotein of the rabies virus against genetic variants of rabies virus isolated worldwide are in progress. Persistent rabies virus infections have been established in several strains of inbred mice. Attempts to isolate infectious virus with an in vitro amplification technique from tissues of these mice have been discouraging. Nonetheless, virus was isolated from either bone marrow or central nervous system tissue of 5/47 (11%) mice which had been infected up to 90 days previously. Experiments to enhance the sensitivity of our detection assay have determined that our in vitro amplification assay is >100 fold more sensitive than detection of mRNA by Northern blot analysis. To increase sensitivity, we are now using RT- PCR. Preliminary results are encouraging in that the ERA rabies virus has been amplified and detected in tissue culture infected cells with both nucleoprotein and glycoprotein primers. Unfortunately, amplification and detection in vitro of the bat street rabies virus which was principally used to establish persistent infections in mice have failed. Presently we are testing nested PCR reactions and new sets of nucleoprotein and glycoprotein primers to detect this virus. Further progress on this project depends on our ability to initially detect in vitro the viruses which were used to establish the in vivo persistent infections. We have determined that both tissue culture adapted and non- tissue culture adapted wild strains of rabies viruses replicate in primary murine macrophages and murine and human macrophage-like cell lines. Persistent infections were established in U937 cells with 2 different strains of virus. Rabies virus replicated in differentiated, but not undifferentiated, HL-60 cells. This data in conjunction with that of the macrophage-like cell lines suggests that more differentiated cells are more permissive to rabies infection. Studies to determine whether microglial cells serve as reservoirs of infection in persistent infections and also contribute to rabies-induced pathology of the brain are in progress.